[show abstract][hide abstract] ABSTRACT: The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8(+) T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such "two-step" inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.
Cancer Immunology and Immunotherapy 04/2008; 57(3):289-302. · 3.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: Allogeneic hematopoetic stem-cell transplantation (alloHSCT) has curative potential for poor risk lymphoma patients due to the graft-versus-lymphoma effect. High non-relapse mortality with conventional high-dose conditioning indicates the necessity for less toxic transplant strategies.
Between 1992 and 1999, 25 patients [median age 37 (20-60) years] with relapsed or refractory non-Hodgkin's lymphoma (NHL, n = 20) or Hodgkin's disease (HD, n = 5) received an alloHSCT in our institution. Patients were grafted from HLA matched (17) or mismatched (2) related, or matched unrelated donors (MUD) (6). NHL histological subtypes were lymphoblastic (6), high grade B/T-cell lymphomas (5), follicular (3), mantle cell (2) and CLL, immunocytic, composite lymphoma and panniculitic T-NHL in one patient each. Patients had received a median of four (range three to six) different therapies before alloHSCT, and 10 patients had relapsed after high-dose chemotherapy and autologous (9) or allogeneic (1) HSCT. Remission status prior to allogeneic SCT was CR1 (1), CR2 (1), relapse (11), partial remission (5) or primary refractory induction failure (7). Conventional myeloablative conditioning (cc) regimens contained total body irradiation 12 Gy (5), busulfan 16 mg/kg (7) or BCNU/VP16 (1). Twelve patients received reduced-intensity conditioning (ric) regimens with fludarabine (FLU) plus alkylating agents. Graft-versus-host disease prophylaxis consisted of cyclosporin A +/- prednisone or methotrexate. Six patients also received anti-T-lymphocyte globulin.
Twenty-four patients engrafted. Best response after alloHSCT was complete remission in 16 of all patients [64%: 95% confidence interval (CI) 44% to 84%] and in 16 of 22 evaluable patients (73%: 95% CI 53% to 93%), partial remission in three of 25 (12%), and no change in three of 25 (12%) patients. Early death prevented response evaluation in three of 25 patients. Non-relapse mortality was 54% (95% CI 15% to 78%) in patients after cc and 17% (95% CI 0% to 41%) after FLU-based ric (P = 0.03). Six patients died due to progressive disease or relapse. Four patients with HD died, three in complete remission due to non-relapse mortality and one with progressive disease. Eleven of 25 patients are alive with a median follow up of 618 days (range 383-2815), with an overall survival of 44% (95% CI 23% to 65%) at 1 year for all patients, while eight of 12 (67%: 95% CI 35% to 98%) patients are alive after ric compared with three of 13 (23%; 95% CI 0% to 50%) after cc (P <0.02).
AlloHSCT induces high rates of complete remission in advanced lymphoma patients, even when the tumor had relapsed after autologous HSCT. It should be considered earlier as part of the therapeutic options in poor risk patients to avoid non-relapse mortality associated with extensive pretreatment. Our novel reduced conditioning regimens show promising results, especially in heavily pretreated patients, and improve survival after allogeneic transplantation.
Annals of Oncology 01/2002; 13(1):135-9. · 7.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Conditioning regimens with reduced intensity are used increasingly for allogeneic stem cell transplantation in elderly or extensively pretreated patients. Two cases of pure red cell aplasia after fludarabine-based conditioning and during immunosuppression with cyclosporin are described. Both patients received ABO-mismatched stem cells and had anti-donor isoagglutinins. Red cell recovery occurred after extended immunosuppression when isoagglutinins had disappeared. Colony assays indicated serologic suppression of the erythrocyte lineage in one patient. Since reduced conditioning permits donor cell engraftment primarily by suppression of host T cells, antibody-mediated immunological complications may occur more frequently than after 'classical' conditioning.
Bone Marrow Transplantation 11/2000; 26(8):911-5. · 3.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: Despite improvements in HLA typing, graft-versus-host disease (GVHD) continues to impair the results after volunteer unrelated donor bone marrow transplantation (VUD-BMT) in adult patients compared with matched sibling BMT. Here, the outcome after VUD-BMT using a specific regimen with high-dose anti-T-lymphocyte globulin (ATG) was analysed. Fifty-five adult patients, median age 34 years (range 17-55 years), with acute or chronic leukaemia or myelodysplastic syndrome (MDS) were transplanted in first complete remission (CR1)/first chronic phase (CP1) (early disease) (n = 21) or in advanced (CR2/CP2, no remission) disease (n = 34) from an unrelated marrow donor. GVHD prophylaxis consisted of ATG-S (Fresenius) 60-90 mg/kg b.w. prior to transplantation, in addition to cyclosporin A and short-course methotrexate. Graft failure did not occur and white blood cell count (WBC) > 1.0 x 10(9)/l was reached at median day +16. The cumulative incidence of acute (a)GVHD grade II-IV was 15% [95% CI (8%, 28%)] and of chronic GVHD was 51% [95% CI (38%, 68%)]. The cumulative incidence of relapse within 1 year was 0% [95% CI (0%, 19%)] and 21% [95% CI (11%, 40%)] for patients with early and advanced disease respectively. With a median follow-up of 28 months (range 16-45 months), 2-year disease-free and overall survival for patients transplanted in CR1/CP1 was 81% and 81% [95% CI (64%, 98%)], respectively, and for patients with advanced disease was 33% [95% CI (17%, 50%)] and 40% [95% CI (23%, 57%)] respectively. Complete and persistent donor chimaerism was seen in 77.5% of 40 patients evaluated. All 14 chronic myeloid leukaemia (CML)-CP1 patients became bcr-abl negative within 250 d. High-dose ATG pretransplant results in a low incidence of severe aGVHD without compromising donor chimaerism or elimination of minimal residual disease. Our results are similar to data obtained after matched sibling donor transplantation.
British Journal of Haematology 10/2000; 111(1):303-13. · 4.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: Between August 1998 and July 1999, 21 patients received a novel protocol of reduced conditioning with fludarabine, carmustine and melphalan (FBM) followed by matched-related allogeneic peripheral blood stem cell transplantation (PBSCT) in a prospective multi-center phase I/II study. Cyclosporin A and 'mini-methotrexate' were used for GVHD prophylaxis. Patients were included because of age, advanced disease, previous transplantation or co-morbidity. Hematopoietic engraftment after allogeneic transplantation was rapid with a median white blood count (WBC) >1 x 10(9)/l on day +11 (range 10-17) and a median platelet count >20 x 10(9)/l on day +13 (range 9-36). Donor chimerism was complete in 16/21 (76%) patients at all time points during follow-up and mixed at least on one occasion in 5/21 (24%) patients. The conditioning regimen was well tolerated with low toxicity even in previously transplanted patients. Thirteen patients (62%) developed acute GVHD grades II-IV. Nineteen out of 21 patients achieved complete (CR, n = 15) or partial remission (PR, n = 4) with a median patient follow-up of 354+ days (range 258-577) for patients alive. The reduced intensity protocol FBM is feasible with rapid engraftment, early achievement of complete donor chimerism, low toxicity, especially in heavily pretreated patients, and good response rates in advanced disease patients.
Bone Marrow Transplantation 08/2000; 26(3):243-50. · 3.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: Because of their hypervariable regions and somatic mutations, the antigen receptor molecules of lymphomas (idiotypes) are tumor-specific antigens and attractive targets for antilymphoma immunotherapy. For the optimal induction of human idiotype-specific cytotoxic T cells (CTL), idiotype was presented to CD8(+) peripheral blood mononuclear cells by monocyte-derived autologous dendritic cells (DC) after the endocytosis of idiotype protein or by idiotype-expressing DC. Recombinant idiotype was obtained as a functionally folded Fab fragment by periplasmic expression in Escherichia coli. Idiotype-expressing DC were generated by transduction with recombinant Semliki forest virus vectors encompassing heavy- or light-chain idiotype genes. Autologous lymphoblastoid cell lines stably transfected with Epstein-Barr virus-based idiotype expression vectors were used as target cells to detect idiotype-specific lysis. CTL stimulated with idiotype-loaded DC showed strong specific, CD8-mediated, and major histocompatibility complex (MHC) class I-restricted cytotoxicity against autologous heavy- and light-chain idiotype. In contrast, stimulation with idiotype-transduced DC resulted in only moderate natural killer cell activity. These data confirm the existence of idiotype-specific CTL in patients with lymphoma, define a "good manufacturing practice"-compatible protocol for the generation of these cells without the requirement of viable lymphoma cells, and favor the processing of exogenous antigen over DC transduction for the induction of MHC I-restricted CTL against idiotypes with unknown antigenicity. (Blood. 2000;95:1342-1349)
[show abstract][hide abstract] ABSTRACT: An expression system for rapid and standardized production of human recombinant immunoglobulin Fab fragments in E. coli was developed. Functional folding of the Fab fragments was accomplished by the dicistronic expression vector pFab.gammakappa containing specialized leader sequences to direct the immunoglobulin heavy and light chains to the periplasmic bacterial space. A C-terminal hexahistidine tag of the Fd chain facilitated metal affinity chromatography and purification to homogeneity as assessed by SDS PAGE and silver staining. Specific antigen recognition by a hybridoma-derived Fab fragment was indistinguishable from that of the corresponding monoclonal antibody. This protocol may be useful for analysis of the antigen specificity of human B cells and for convenient production of lymphoma-derived idiotype protein for vaccination strategies. To obtain unmodified immunoglobulin cDNA sequences from small human biopsies for insertion into pFab.gammakappa, oligo(dG)-tailed cDNA was amplified with an oligo(dC)- and nested mu or kappa constant region-specific primers. Using single sets of primers for each class of immunoglobulin transcripts, the products of this anchored PCR reflected the relative abundance of the starting cell population and permitted reliable identification of clonal, lymphoma-derived sequences for subsequent expression cloning.
Journal of Immunological Methods 11/1999; 229(1-2):141-53. · 2.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human melanoma cell lines may induce a specific T cell response against tumor cells in vitro. However, after repeated restimulation with autologous tumor cells, expansion of CTL is limited and often apoptosis of the T cells occurs. In order to improve conditions inducing primary T cell responses and thus allowing further expansion of tumor specific T cells for an adoptive transfer, we transfected human melanoma cells with the B7.1 gene (CD80), known to be a potent costimulatory molecule for T cell activation. CD80 expression on melanoma cells resulted in improved primary T cell activation, especially of CD8+ T cells. Furthermore, restimulation with CD80+ tumor cells gave rise to long term proliferating CD8+ T cell lines demonstrating an 100-fold expansion of T cells compared to the 20-30-fold increased numbers obtained with the controls (parental tumor cells +/- anti-CD28). T cells stimulated with CD80+ melanoma cells were found to display a MHC class I-restricted cytotoxic activity against the autologous tumor cells. In conclusion, these studies demonstrate the requirement of costimulation in generating large numbers of tumor specific T cells in vitro that may be used for an adoptive transfer in tumor immunotherapy.
European cytokine network 10/1999; 10(3):329-36. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Die Gentherapie in der Onkologie verfolgt derzeit Ansätze, die ihre therapeutische Wirkung entweder durch eine direkte Korrektur
genetischer Defekte in Tumorzellen erreichen, eine antiproliferative Wirkung, Apoptose oder Suizid in Tumorzellen auslösen,
eine Immunantwort gegen Tumorzellen induzieren oder konventionelle Therapieverfahren komplementieren.
Der Onkologe 09/1999; 5(10):898-909. · 0.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human lymphocytes remain among the most promising target cells for gene therapy. Gene-modified lymphocytes have been used successfully to treat adenosine deaminase (ADA)-deficient patients and to control GvHD after allogeneic BMT. Because activation and proliferation of T cells are necessary for efficient retrovirus-mediated gene transfer and subsequent selection of transduced cells, mononuclear cells (MNC) from steady-state and G-CSF-stimulated peripheral blood were activated by short exposure to the mitogen PHA, the anti-CD3 antibody OKT3, or both in the presence of different concentrations of recombinant IL-2. Using OKT3 (10 or 30 ng/ml) and IL-2 (100 U/ml), T cells expanded efficiently during a 14-day culture period. Cell expansion was similar under serum-free conditions. The immunophenotypic profile over time showed a marked increase in CD8+ cells, leading to a reversed CD4/CD8 ratio of 1:2 and a slight increase in CD56+ cells. Supernatant-based centrifugal transduction of primary human T lymphocytes was compared with supernatant transduction on the extracellular matrix protein fibronectin. Transduction with cell-free retrovirus-containing supernatant in tissue culture flasks coated with human plasma fibronectin led to significantly higher transduction efficiencies (20% +/- 7.5%) than centrifugal transduction in uncoated culture flasks (13.6% +/- 5.1%)(p = 0.041). To both rapidly characterize transduced cells and isolate these from residual nontransduced but biologically equivalent cells, an amphotropic Moloney murine leukemia virus (MoMuLV)-based retroviral vector containing the intracytoplasmically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene was used. FACS sorting of T cells after transduction resulted in >90% LNGFR+ cells and was much faster than enrichment of transduced cells through growth in G418-selection medium. These results show that supernatant-based retroviral gene transfer into primary human T lymphocytes can be enhanced by fibronectin. Ectopic expression of a cell surface protein can be used to rapidly and conveniently quantitate transduction efficiency through FACS analysis and to efficiently enrich transduced cells through FACS sorting.
Journal of Hematotherapy & Stem Cell Research 09/1999; 8(4):401-10.
[show abstract][hide abstract] ABSTRACT: The objectives of this study were to assess cytokine secretion in human renal cell carcinoma (RCC) and to identify cytokines contributing to the immunomodulatory effect of tumor cells.
Cytokine secretion in the supernatant of primary tumor cell cultures (PTCC) and corresponding cell lines (CL) was assayed using ELISA. Tumor cells were characterized by morphology, immunocytochemistry, and flow-cytometric analysis. Tumor-cell-induced T cell activation was determined by coculture of gamma delta and alpha beta T cell clones with tumor CL.
We assessed the cytokine secretion of tumor cells from 27 PTCC and their corresponding CL (3/27) of RCC. We found that RCC predominantly produced both pro-inflammatory and T-cell-inhibitory cytokines, such as IL-8, IL-6, GM-CSF, TNF-alpha, IL-10 and TGF-beta 1. CL were adapted to serum-free medium which may prove as a useful tool in future studies of cytokine secretion in RCC. In addition, we used gamma delta and alpha beta T cell clones to assess the immunomodulatory effect of tumor cells from RCC and found that predominantly gamma delta T cells were activated by RCC.
Our data suggest that RCC produce large amounts of both pro-inflammatory and T-cell-inhibitory cytokines that potentially could influence the immune response of the host, especially tumor-specific cytotoxic T cells.
European Urology 02/1999; 35(1):70-80. · 10.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: A 46-year-old female presented with acute myeloid leukemia during complete remission of multiple myeloma after extensive treatment with alkylating agents. Leukemic blasts expressed CD34, platelet esterase and gp IIIa. RT-PCR analyses of peripheral blood cells detected a p190 type BCR-ABL rearrangement and high levels of MDR1. The patient expired during neutropenia shortly after induction chemotherapy. Autopsy revealed persistent blasts in the bone marrow, spleen and liver. 'Secondary' acute myeloid leukemia with megakaryoblastic features and p190-type BCR-ABL rearrangement has not previously been reported. The possibility that the combination of a BCR-ABL rearrangement with overexpression of MDR1 may have contributed to the treatment-refractory course is discussed.
Leukemia Research 12/1998; 22(11):1021-7. · 2.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: Success of gene replacement therapy depends on long-term, high level expression of the transgene. Gene therapy vectors incorporating a promoter of a constitutively active eukaryotic gene may allow long-term expression in vivo, but the expression level may be insufficient for therapeutic effects. To enhance transcription from eukaryotic promoters, a strategy with dicistronic vectors encoding the therapeutic gene of interest together with a transcription factor that binds and activates the promoter was tested. Expression vectors for the chimeric tet repressor/VP16 transcription factor (tTA) driven by the human beta-actin promoter were constructed, and tandem tet operators were inserted within the promoter. This arrangement significantly enhanced expression of G-CSF in fibroblasts to higher levels than the immediate/early CMV promoter. Stably transfected fibroblast clones produced up to 2.4 microg G-CSF per 10(6) cells x 24 h. After injection of genetically engineered cells into SCID mice, the enhanced beta-actin promoter construct resulted in marked leukocytosis, whereas the unmodified promoter had only a marginal therapeutic effect. Transcription factor-enhanced, feed-back-activated human promoters may thus achieve higher expression levels than viral control elements, and may be advantageous for gene therapy due to high constitutive activity in vivo.
International Journal of Molecular Medicine 11/1998; 2(4):423-8. · 1.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Epithelia-associated dendritic cells (DC) including Langerhans cells in the skin (LC) are precursors of lymph node located interdigitating DC (iDC). CD1a+ LC are known to be derived from CD34+ haemopoietic progenitor cells (HPC); however, cells of an intermediate differentiation state that are CD34- and CD1a- have not been identified. Monitoring the differentiation pathway of HPC in the presence of GM-CSF+IL-4, we observed the emergence of a distinct LC precursor population that was CD33+ CD13+ CD4+ CD38+ CD44+ CD34- CD14- CD1a-. The cells could be separated by FACS due to a unique CD44/CD38 expression pattern or by CD44 expression in conjunction with the SSC profile. It was found that they were similarly generated in the presence of GM-CSF alone and were detectable in culture for at least a week. Irrespective of being generated in the presence of GM-CSF+IL-4 or GM-CSF alone, CD44/SSC-sorted precursor cells matured to MHC class II compartments (MIIC) and Birbeck granules (BG) expressing LC, when subsequently cultured in the presence of GM-CSF+IL-4. When IL-4 was omitted, however, the same cells matured to phagocytically active adherent macrophages (Mphi). These culture conditions were associated with a > 4-fold increase in the concentration of IL-6 when compared to those used for LC differentiation. The identification of a distinct oligopotent precursor cell population that can deliberately be induced to give rise to BG+ MIIC+ CD1a+ CD14- LC or to adherent CD14+ Mk further substantiates the close relationship of monocytes and DC and may help to identify its in vivo equivalent.
British Journal of Haematology 06/1998; 101(2):231-41. · 4.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dendritic cells (DC) have been generated in vitro from either CD34+ haemopoietic progenitor cells (HPC) or peripheral blood monocytes (Mo) in the presence of specific cytokine combinations, including granulocyte-macrophage colony-stimulating factor (GM-CSF). Since differences between DC from either source may be important for the clinical use of these antigen-presenting cells (APC), a comparative analysis was performed. HPC were expanded in the presence of interleukin (IL)-3, IL-6 and stem cell factor (SCF) (days 1-7) and subsequently induced by IL-4+ GM-CSF (days 8-26) to differentiate to Langerhans-type cells (pLC). The latter cytokines were similarly used to generate Mo-derived LC (mLC). Maturation of both cell types, pLC and mLC, to interdigitating DC-type cells (iDC) was induced by tumour necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS). Analysis of mLC/pLC and miDC/piDC with respect to morphology, phenotype, antigen uptake and presentation revealed a high similarity of DC from either source. The majority of mLC, however, exhibited a more mature differentiation stage, compared to pLC, evidenced from lower numbers of multilaminar MHC class II compartments and less efficient APC function for extracellular protein antigens. Although macropinocytosis was performed by LC, neither LC nor iDC from either source were able to take up > or = 0.5 microm latex beads. However, phagocytosis of 0.5 microm and 1 microm beads was performed by Mo that could subsequently be induced to become iDC, thus providing the unique opportunity to present phagocytosed material in DC-type fashion. Mo may be the preferential source for clinical use of iDC-type cells since preparation and culture are easier to perform and are less costly while APC function is similar to HPC-derived iDC.
British Journal of Haematology 12/1997; 99(3):490-9. · 4.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: In a phase I trial designed to study a vaccine composed of autologous tumor cells and interleukin-2 gene transfected fibroblasts we analyzed lymphocytes infiltrating the vaccination site (VIL) in two melanoma patients. Functional studies demonstrated that numbers of MHC class I restricted cytotoxic T cells directed against the autologous tumor had increased at the immunization site in both cases. Analysis of the variability of T cell receptors (TCR) in the VIL of one patient revealed that the cytotoxic T lymphocytes consisted of a predominant population of TCRBV21S3+ T cells. Enrichment of this subpopulation to more than 99% by specific anti-TCRBV21S3 monoclonal antibody linked immunomagnetic beads and sequencing of the TCR-beta chain disclosed exactly the same V-D-J junctional sequence in all eight TCRBV21 transcripts from these VIL. The identical sequence was also detected in all eight TCRBV21 transcripts from the patient's tumor-infiltrating lymphocytes, indicating that the same CTL clone had infiltrated the tumor, circulated in the peripheral blood, and was amplified at the vaccination site. The TCRBV21S3+ T cells were also found to display an MHC class I restricted cytotoxic activity specifically directed against the autologous tumor cells. At the beginning of treatment these cells were undetectable at the vaccination site and delayed-type hypersensitivity testing was negative, contrasting with the positive results after therapy. Thus it is likely that vaccination with autologous tumor cells plus interleukin-2 gene transfected allogeneic fibroblasts had induced not only local accumulation but also an increase in the frequency of circulating tumor specific CTL.
Journal of Molecular Medicine 05/1997; 75(4):290-6. · 4.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: We evaluated the survival, transgene production, and copy numbers of integrated plasmid units per host genome after lipofection with mono- and bicistronic plasmid vectors in different cell lines and under various conditions. The addition of an integration enhancing murine sequence nontranscribed spacer (NTS) to the plasmids increased transfection efficiency, survival, and transgene expression. However, in human fibroblast cells this sequence had only marginal effects on overall plasmid copy number in bulk cultures. Clones producing the highest amounts of the transgene contained only one or two copies of plasmid per genome, independent of cell type and plasmid design.
Journal of Molecular Medicine 04/1997; 75(3):223-9. · 4.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vaccination with gene-transfected tumor cells has recently been proposed as a new strategy in the immunotherapy of cancer. Since autologous tumor cells provide an optimal antigen profile, the possibility of generating single cell suspensions from renal cell carcinoma (RCC), malignant melanoma (MM), colon carcinoma (CC), and non-small-cell lung cancer (NSCLC) biopsies was investigated. One hundred and seventy-four tumor biopsies were processed by mechanic and enzymatic dissociation, yielding 1-2 x 10(6) cells/g tumor (median), irrespective of tumor type. Primary tumor cell cultures (PTCC) of > or = 10(7) cells were established from 29 of 86 (34%) RCC, 14 of 38 (37%) MM, 11 of 23 (48%) NSCLC and 4 of 27 (15%) CC specimens. The amount of non-tumor cells, as assessed by morphology and immunocytology, was generally low (< 30%) in RCC (35 of 41) and MM (11 of 17), while it exceeded 60% in 8 of 11 PTCC from NSCLC and 3 of 11 CC. A high tumor cell yield was obtained in biopsies with a high degree of vascularization and in the virtual absence of necrosis. Thus, PTCC > or = 10(7) cells were obtained in 73% of MM with a high degree of vascularization and in 22% of MM with a low degree of vascularization (p < 0.007). Long-term tumor cell cultures exceeding 20 passages were established in 24 of 86 (18%) RCC, 7 of 38 (18%) MM and 3 of 27 (11%) CC, while successful implantation in nude mice was achieved in 8 of 20 RCC and 5 of 10 MM. Thus, under the conditions described, > or = 10(7) primary tumor cells of high purity could be generated from about one third of RCC and MM biopsies, while the success rate increased to > 50 and > 70%, respectively, in samples with a high degree of vascularization generated by an optimized biopsy technique excluding necrotic parts.
European Surgical Research 01/1997; 29(4):292-302. · 0.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: Tumor cells transfected to express immunostimulatory cytokines, or admixed with similarly modified bystander cells, are able to induce immune responses against unmodified tumor cells in animal models. For treatment of human patients, a vaccine composed of autologous tumor cells and IL-2-secreting allogeneic fibroblasts was developed. Autologous tumor cells were isolated from biopsy specimens. A clone (KMST 6.14) of an immortalized human fibroblast line that stably secreted 5290 IU IL-2 per 10(6) cells and per 24 hr was obtained by cationic lipofection with an expression construct for human IL-2 and Neo(r). Fifteen patients with refractory malignant tumors received 3-4 injections of irradiated KMST6.14 and autologous tumor cells in a phase-I clinical trial. Increasing transient inflammatory responses without systemic toxicity developed at vaccination sites and after injections with irradiated tumor cells only (p < 0.05). These sites contained a dense infiltrate of CD3+ T cells with numbers of CD4+ helper cells exceeding those of CD8+ cytotoxic T cells (CTL). CD8+ T-cell lines isolated from vaccination sites of 2 malignant melanoma patients but not of renal-cell carcinoma patients exhibited a dominant lytic activity against autologous tumor cells in vitro. CD8+ T-cell clones established from the vaccination site of 1 of 2 renal-cell carcinoma patients preferentially lysed autologous and partially matched allogeneic renal-cell carcinoma cells. In conclusion, a vaccine composed of IL-2 gene-transfected allogeneic fibroblasts and autologous tumor cells is able to enhance specific anti-tumor T-cell responses in vivo without major side-effects. Malignant melanoma and renal-cell carcinoma appear to be promising entities for testing of similar approaches in future therapeutic trials.
International Journal of Cancer 01/1997; 70(3):269-77. · 6.20 Impact Factor